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The use of RNA interference as a tool to examine gene function, and its potential as a species-specific pesticide in the yellow fever mosquito, Aedes aegyptiSingh, Aditi Diana 06 April 2011 (has links)
RNA interference (RNAi) is a gene silencing mechanism induced by double-stranded RNA (dsRNA). RNAi has been used extensively to create loss-of-function mutants in many species to identify the functions of genes, but it also has the potential to be used as a species-specific pesticide if the dsRNA can silence essential genes in pests. The mosquito Aedes aegypti is a vector of numerous viruses including Dengue and West Nile virus, and is frequently controlled by chemical insecticides. With growing concerns about the extensive use of broad-spectrum pesticides, new control methods are eagerly sought. In this study, I examined the efficacy of feeding pesticidal dsRNAs to mosquito larvae. A dose-dependent RNAi response and mortality was observed when larvae were fed dsRNA targeting several different genes. Unlike RNAi in the related dipteran Drosophila melanogaster, RNAi in A. aegypti also appeared to be systemic, spreading beyond the gut to other tissues. A degree of species-specificity was also observed, as dsRNA specific to the D. melanogaster β-tubulin gene killed D. melanogaster larvae but did not kill mosquito larvae.
RNAi was also used to determine the function of a newly-identified A. aegypti cytochrome P450 (CYP) gene, Aacyp. This gene showed male-biased expression in the mosquitoes, and was expressed primarily in the male abdomen and/or thorax, but unlike some other insect male-biased CYPs, Aacyp was not highly expressed in the reproductive structures. While dsRNA injections successfully knocked down expression of Aacyp, no discernable change in reproductive or male-specific behaviours were noted. Nevertheless, RNAi is still considered a highly versatile tool for both gene function studies and has promising potential to be developed into a novel class of pesticides.
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The use of RNA interference as a tool to examine gene function, and its potential as a species-specific pesticide in the yellow fever mosquito, Aedes aegyptiSingh, Aditi Diana 06 April 2011 (has links)
RNA interference (RNAi) is a gene silencing mechanism induced by double-stranded RNA (dsRNA). RNAi has been used extensively to create loss-of-function mutants in many species to identify the functions of genes, but it also has the potential to be used as a species-specific pesticide if the dsRNA can silence essential genes in pests. The mosquito Aedes aegypti is a vector of numerous viruses including Dengue and West Nile virus, and is frequently controlled by chemical insecticides. With growing concerns about the extensive use of broad-spectrum pesticides, new control methods are eagerly sought. In this study, I examined the efficacy of feeding pesticidal dsRNAs to mosquito larvae. A dose-dependent RNAi response and mortality was observed when larvae were fed dsRNA targeting several different genes. Unlike RNAi in the related dipteran Drosophila melanogaster, RNAi in A. aegypti also appeared to be systemic, spreading beyond the gut to other tissues. A degree of species-specificity was also observed, as dsRNA specific to the D. melanogaster β-tubulin gene killed D. melanogaster larvae but did not kill mosquito larvae.
RNAi was also used to determine the function of a newly-identified A. aegypti cytochrome P450 (CYP) gene, Aacyp. This gene showed male-biased expression in the mosquitoes, and was expressed primarily in the male abdomen and/or thorax, but unlike some other insect male-biased CYPs, Aacyp was not highly expressed in the reproductive structures. While dsRNA injections successfully knocked down expression of Aacyp, no discernable change in reproductive or male-specific behaviours were noted. Nevertheless, RNAi is still considered a highly versatile tool for both gene function studies and has promising potential to be developed into a novel class of pesticides.
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RNA interference of Bmp-4 gene expression and midface development in postimplantation mouse embryos a thesis submitted in partial fulfillment ... for the degree of Master of Science in Orthodontics ... /Shuman, Jerome B. January 2005 (has links)
Thesis (M.S.)--University of Michigan, 2005. / Includes bibliographical references.
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Identification and functional characterization of mosquito genes that affect Plasmodium developmentJaramillo-Gutierrez, Giovanna 07 October 2009 (has links)
Les moustiques anophèles sont les vecteurs du parasite Plasmodium l’agent du paludisme. Le parasite subit des pertes massives pendant son cycle de développement chez l’anophèle, ce qui suggère que les moustiques sont capables de développer une réaction immunitaire efficace contre le parasite. La connaissance de l’immunité et de la résistance des moustiques au genre Plasmodium provient principalement de systèmes de laboratoire qui utilisent des espèces de parasites de rongeurs ou d’oiseaux comme modèles du paludisme humain. Les observations présentées dans cette thèse suggèrent que certains gènes comme Tep1 et LRIM1 sont des médiateurs de réponses antiparsitiques contre différents Plasmodiums dans différents vecteurs. Cependant, le degré d'efficacité avec laquelle un moustique est capable de réduire le nombre de parasites peut être variable surtout entre combinaison de souche de moustique et de souche de parasite différentes, selon que la paire soit hautement compatible ou non.
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Construction of adenovirus vectors for studies of protein function and RNA interference /Berenjian, Saideh, January 2006 (has links)
Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.
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Light activated RNA interferenceShah, Samit, Friedman, Simon H. January 2007 (has links)
Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2007. / "A dissertation in pharmaceutical science and chemistry." Advisor: Simon H. Friedman. Typescript. Vita. Description based on contents viewed July 16, 2008; title from "catalog record" of the print edition. Includes bibliographical references (leaves 206-220). Online version of the print edition.
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Expression of anti-HBV primary micro-RNA shuttles using an inducible promoter system.Mlambo, Tafadzwa 28 March 2014 (has links)
Hepatitis B virus (HBV) infection is an important global health concern and chronic carriers of the virus are at high risk of developing hepatocellular carcinoma (HCC) and cirrhosis. Current therapies are only partially effective, which emphasises the need for improved treatment strategies. Harnessing the RNA interference (RNAi) pathway as a treatment strategy against HBV has shown great promise. However, there are obstacles that need to be overcome before RNAi-based treatment of HBV infection is realised. These include problems of liver tissue targeting and dose regulation. This study investigated the use of a liver specific and mifepristone-inducible RNA polymerase (Pol) II promoter system for the specific and precise regulation of anti-HBV sequence expression. The inducible system used consists of two expression cassettes; one containing the
regulator/transactivator protein and another containing the transgene. Natural primary microRNA (pri-miR) mimics, pri-miR-31/5 and pri-miR-31/5/8/9, were used as anti-HBV sequences. Firefly luciferase gene expression was used to test modulation by the inducible system and to determine optimal induction conditions. The pri-miR-31/5, pri-miR-31/5/8/9 and luciferase encoding fragments were incorporated into the plasmid vector pRS17 that bears the inducible promoter, creating pRS-31/5, pRS-31/5/8/9 and pRS-Luc respectively. Firefly luciferase expression with this system was shown to be inducible and mifepristone dose-dependent. Effective knockdown of HBV gene expression was achieved with both pRS-31/5 and pRS-31/5/8/9 in vitro and in vivo. However, with high vector amounts, similar efficiency in silencing of HBV gene expression was observed in the presence and absence of the inducer mifepristone suggesting leaky expression of the pri-miRs. To confirm this, knockdown studies were carried out with the pri-miR-31/5/8/9-expressing cassette separated from the transactivator cassette. HBV gene expression knockdown was observed with the pri-miR-31/5/8/9 cassette alone confirming leaky expression from the inducible system. Leakiness appears to be as a result of the E1B promoter driving the expression of the pri-miRs in the absence of mifepristone. However, reducing the vector amounts decreased basal expression and improved the inducibility of the system in cell culture studies. Successful propagation of an inducible and liver-specific RNAi-activating expression system will address the difficulty of achieving dose control of RNAi effectors and contribute to advancing the use of RNAi for HBV treatment.
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Genetic factors affecting the RNA interference pathway of Aedes aegypti mosquitoesHaac, Mary Etna Richter 30 December 2013 (has links)
Aedes aegypti mosquitoes are the vectors of many significant arboviruses that cause tremendous social and economic impact. RNA interference (RNAi) plays a crucial role in the vector competence of mosquitoes and is often targeted in studies involving mosquito innate immunity, genetics-based vector control strategies, and the development of viral-resistant transgenic mosquitoes. In general, RNA interference is induced by double stranded RNA (dsRNA) and results in the inhibition of cognate gene expression. There are several different RNA interference pathways, with distinct functions and mechanisms. The micro RNA pathway is important for endogenous gene regulation and development. The endogenous small interfering RNA (endo-siRNA) pathway functions in gene regulation and protection of the genome from the deleterious effects of transposable elements. The exogenous siRNA (exosiRNA) pathway is a major contributor to mosquito innate immunity and vector competence by limiting viral replication during infection. Lastly, the piwi RNA (piRNA) pathway primarily functions in protecting the genome from the deleterious effects of transposable elements.
While the structure and function of many genes involved in Drosophila RNAi have been characterized, the corresponding mosquito orthologs have only been peripherally described or remain unknown. Thus, the overall purpose of this study is to improve the understanding of mosquito RNAi mechanisms by identifying and analyzing genetic factors involved in the various pathways. This research especially focuses on characterizing and analyzing putative doubleiii stranded RNA binding proteins (dsRBPs) important to the function of the RNAi initiator and effector complexes. Two genes, r2d2 and r3d1 are orthologs of Drosophila genes known to have important roles in the RNAi initiator complex. A third member of the same family, which we refer to as extra loquacious (exloqs), appears to have no known orthologs outside of the Aedes genus. Structural characterization of these genes included identification of single nucleotide polymorphisms (SNPs), novel exons and alternative splice variants. RT-PCR assays were utilized to examine differential expression of all three genes in specific tissues and developmental stages. Sub-cellular fractionation assays enabled intracellular localization of the RNAi proteins within Ae. aegypti cells. Co-immunoprecipitation of tagged dsRBPs revealed protein-protein interactions between specific dsRBPs and known RNAi factors. In addition, an exo-siRNA sensor was designed and tested in-vivo and in-vitro with the purpose of facilitating the identification of novel genetic factors involved in this anti-viral pathway. Lastly, TALENbased gene disruption was successfully employed to knockout the exloqs gene in Ae. aegypti mosquitoes, enabling further analysis into the function of this gene. The research described in this document provides further insight into mosquito innate immunity and gene regulation, which is important to the advancement of genetics-based vector control strategies. / Ph. D.
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Screening for genes involved in cilia formation and functionHall, Emma Andisi January 2012 (has links)
Cilia are small microtubule based structures found on the surface of almost all mammalian cells, enclosed in a highly specialised extension of the cell membrane. Components of several key developmental signalling pathways, in particular Hedgehog (Hh) signalling, are enriched in cilia and cells with mutations in cilia structure show aberrant signalling, suggesting cilia act as “antennae” to focus these signalling cascades. A spectrum of human diseases, termed ciliopathies, are caused by problems in cilia formation or cilia function, which display wide ranging phenotypes from embryonic lethality to retinal degeneration, polydactyly to cystic kidneys. Despite recent advances in the understanding of the essential roles cilia play in mammalian development, exactly how these complex structures are put together, how they carry out their diverse functions, and how they are regulated is not well understood. In this thesis, I describe a screen for genes involved in cilia formation and function. While optimising ciliogenesis and immunofluorescence protocols for the screen, the phenotypes of two ciliary mutant cell lines were analysed. Wdr35yet/yet and Dync2h1pol/pol mouse lines were identified in an ENU screen for genes involved in early development, and shown to have gross phenotypes similar to other ciliary mutants (Mill et al. 2011). Intraflagellar transport (IFT) is the active transport of proteins up and down the ciliary axoneme. Dync2h1 is a retrograde IFT motor component, whereas Wdr35 is part of the retrograde IFT-A complex. In this thesis, the cellular phenotypes of mouse embryonic fibroblasts derived from these mutants are described, showing that despite the fact both genes are thought to be involved in retrograde IFT, they show distinct ciliary phenotypes, suggesting novel roles for Wdr35 in mouse ciliogenesis. An siRNA screen was carried out in mouse fibroblasts to identify genes involved in (i) cilia formation, assayed by immunofluorescence for ciliary markers, and (ii) cilia function, assayed by activity of a Hh responsive luciferase transgene as an indirect readout of ciliary function. Although scalable, I initially screened a small test set of thirty-six putative cilia candidates, identified by cross species transcriptomic analysis. We identified several possible hits, many of which were in the ciliome database but also importantly, several genes with no known link to ciliogenesis. Repeats, correlation of phenotype to knockdown efficiencies and localisation studies validated two hits, Ccdc63 and Azi1. Ccdc63 is a novel coiled-coil gene with no previous link to ciliogenesis; the phenotype for this gene was analysed in real time using fluorescently tagged ciliary markers. A second hit, Azi1, was followed up in more detail. The reduction in ciliogenesis upon Azi1 knockdown was confirmed with separate siRNAs, and was rescued by overexpressing siRNA insensitive Azi1-GFP, confirming the phenotype is not due to off-target effects of the siRNAs. Azi1 gene trap mutant mice were generated and confirmed to be null mutations. Surprisingly, the mice survive, showing Azi1 is not essential for mammalian ciliogenesis. However, mutant males are infertile, with highly reduced sperm count and sperm abnormalities indicative of an arrest at Stage IX of spermiogenesis, when the flagellum, a highly specialised motile cilium, forms. The small number of sperm that do get to the epididymus are immotile. We suggest Azi1 is essential to in the formation of the sperm flagella and male fertility.
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Optimisation of expressed RNA interference effecters for the inhibition of hepatitis B virus ereplicationEly, Abdullah 23 February 2010 (has links)
PhD, Faculty of Health Sciences, University of the Witwatersrand, 2009 / Chronic infection with the hepatitis B virus (HBV) is a major risk factor for
cirrhosis and hepatocellular carcinoma, which is the sixth most common cancer worldwide.
Available treatment for chronic HBV infection has limited efficacy in preventing
associated complications. The compact and multifunctional nature of the viral genome
limits its mutability making HBV an ideal candidate for therapy based on nucleic acid
hybridisation. The potent and specific gene silencing that can be achieved with RNA
interference (RNAi) has fueled interest in exploiting this pathway as a therapeutic
modality. Synthetic and expressed RNA sequences have been used to activate RNAi.
These engineered sequences mimic natural substrates of the RNAi pathway, which allows
them to enter and reprogramme the pathway to effect silencing of intended targets.
Tradionally expressed RNAi activators have been transcribed as short hairpin RNA
(shRNA) sequences from RNA polymerase III (Pol III) promoters. These shRNA mimic
precursor microRNA (pre-miRNA) and consequently enter the RNAi pathway at a
relatively late stage. Overexpression of shRNA sequences from Pol III promoters,
specifically the U6 promoter, has been associated with toxic side effects and has raised
concerns about the use of expressed RNAi activators. Another concern of developing
therapeutic RNAi expression cassettes is the emergence of HBV mutants that are resistant
to silencing by a single expressed RNAi effecter. These points have highlighted the need
for the development expressed RNAi activators that are effective at low concentrations and
capable of combinatorial silencing. To address these issues the aim of this study was to
assess the feasibility of anti HBV effecter sequences that mimic an early substrate (viz.
primary miRNA or pri-miRNA) of the RNAi pathway. Pri-miRNA expression is typically
under the transcriptional control of Pol II promoters. Consequently RNAi activators that
Abstract - xi -
mimic pri-miRNA, so-called pri-miR shuttles, may be expressed from Pol II promoters.
Initially a panel of shRNA expression cassettes driven by a Pol III promoter was
constructed and silencing of HBV replication assessed. Pri-miR shuttles were then
designed by incorporating guide sequences of the most effective anti HBV U6 shRNA into
naturally occurring pri-miR-122 and pri-miR-31. Potent inhibition of viral replication was
observed with both Pol III and Pol II-driven pri-miR shuttle expression cassettes in vitro
and in vivo. Subsequently liver-specific pri-miR-122 and multimeric pri-miR-31 shuttle
expression cassettes were created. Pri-miR-122 shuttle sequences expressed from the
alpha-1 antitrypsin promoter and HBV basic core promoter exhibited the best liver-specific
silencing. Polycistronic pri-miR-31 shuttle sequences were shown to produce multiple
RNAi activators capable of silencing multiple target sequences. Silencing by the pri-miR
shuttle sequences was independent of toxic effects that arise from induction of the
interferon response or saturation of the endogenous miRNA pathway. Pri-miR shuttles
clearly represent an improved option for the use of expressed shRNA and brings
therapeutic RNAi technology a step closer to clinical application.
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